Intratumoral vein-formation promoting agent

ABSTRACT

Provided are agents comprising a phosphatidylcholine as an active ingredient to serve as a vein-formation promoting agent capable of promoting vein-like morphological change of tumor vessels, a vessel-diameter enlarging agent capable of enlarging the diameter of tumor vessels, a blood vessel-connection promoting agent capable of promoting connection of tumor vessels to each other without mediation of a lysophospholipid receptor, a leukocyte-infiltration promoting agent capable of promoting infiltration of leukocytes throughout a tumor region without mediation of a lysophospholipid receptor, and an antitumor immunostimulatory agent capable of promoting infiltration of leukocytes throughout a tumor region without mediation of a lysophospholipid receptor.

TECHNICAL FIELD

The present disclosure relates to a vein-formation promoting agent, avessel-diameter enlarging agent, a blood vessel-connection promotingagent, a leukocyte-infiltration promoting agent and an antitumorimmunostimulatory agent each comprising a phosphatidylcholine as anactive ingredient.

BACKGROUND ART

The formation of new blood vessels in normal tissue takes place via theprocess of vasculogenesis and a new circulation network is established.The process of vasculogenesis includes the steps of development ofvascular endothelial cells, assembly of the endothelial cells intotubular structures (tubulogenesis), and vascular maturation by muralcell coverage of the endothelial cells. On the other hand, inflammation-or hypoxia-induced formation of new blood vessels from preexisting bloodvessels takes place via the process of angiogenesis (sprouting bloodvessel formation). The formation of new blood vessels in tumors alsotakes place via the process of angiogenesis. Such tumorneovascularization makes it possible to supply tumor cells with oxygenand nutrients. Therefore, antitumor therapies have been developedfocusing on tumor angiogenesis inhibition to inhibit tumor growth.

In 1971, a tumor-secreted factor was found to induce the formation ofnew tumor vessels from preexisting blood vessels (Non Patent Literature1). This angiogenic factor was identified as vascular endothelial growthfactor (VEGF). VEGF plays a role in vascular endothelial cell growth andtubulogenesis by activating VEGF receptors (VEGFR1, 2, 3) expressed invascular endothelial cells, in particular VEGFR2. The first developedanti-VEGF drug is an anti-VEGF neutralizing antibody, and this antibodyhas been clinically used early on as an angiogenesis inhibitor (NonPatent Literature 2). However, anti-VEGF neutralizing antibodies havebeen proven not to produce antitumor effect when used alone. Similarly,VEGF receptor tyrosine kinase inhibitors, which are a different type ofangiogenesis inhibitor developed after anti-VEGF neutralizingantibodies, have been proven not to produce antitumor effect when usedalone. On the other hand, a combined use of such an angiogenesisinhibitor and an anticancer drug has been clinically proven to produce asuperior effect as compared with the use of the anticancer drug alone.Recent basic medical studies have indicated that the therapeutic effectof the combined use of an angiogenesis inhibitor and an anticancer drugis attributed to partial normalization of tumor vessels by theangiogenesis inhibitor and consequent improvement of intratumoraldelivery of the anticancer drug (Non Patent Literature 3).

The lumina of normal blood vessels are structurally stabilized byadhesion of mural cells to vascular endothelial cells. Individualvascular endothelial cells tightly adhere to each other via variousadhesion molecules, including VE-cadherin, claudin 5, integrins andconnexins, and this structure contributes to the control of the passageof substances and cells from the blood vessels to prevent their massiveleakage. Further, adherens junctions are formed between vascularendothelial cells and mural cells in normal blood vessels and serve tocontrol vascular permeability by limited molecular transport betweenvascular endothelial cells and mural cells. Normal blood vessels on theright and left sides run parallel to one another. On the other hand,tumor vessels have various abnormalities. Specifically, intratumoralvessels are hyperpermeable, tortuos, dilated, irregularly branched andpartially saccular. In addition, vascular endothelial cells ofintratumoral vessels are also morphologically abnormal. Further, muralcells for covering vascular endothelial cells are highly interspersedand weakly adhere to vascular endothelial cells in the central region ofa tumor, and such mural cell coverage is even absent in most part of thevessel. These abnormalities are mainly caused by over-secretion of VEGFin tumors.

VEGF is a potent growth factor for vascular endothelial cells and servesto inhibit cell-cell adhesion in vascular endothelial cells, therebyincreasing vascular permeability. When such an increased vascularpermeability continues, serum components and fibroblasts accumulate inthe deep part of a tumor and then the interstitial pressure thereinsignificantly increases. As a result, the intraluminal pressure in bloodvessels becomes equal to the interstitial pressure in the deep part ofthe tumor, and this condition impedes the delivery of drugs and the likefrom blood vessels to the tumor tissue. Moreover, the influx of immunecells, such as lymphocytes, into the tumor is also inhibited. This makesit impossible to induce cancer cell death. Once the intracellularsignaling of VEGF is blocked, cell-cell adhesion in vascular endothelialcells is restored, and increased vascular permeability returns tonormal. As a result, the intraluminal pressure in blood vessels becomeshigher than the interstitial pressure in the deep part of the tumor,thus providing an environment allowing anticancer drug delivery andimmune cell influx from blood vessels to the tumor tissue. Therefore, acombined use of an angiogenesis inhibitor and an anticancer drug isconsidered to produce a superior effect as compared with the use of theanticancer drug alone. In addition, due to the improved influx of immunecells, cancer shrinkage may be achieved even without use of anticancerdrugs.

Based on this hypothesis, the normalization of vascular permeability intumors for induction of drug delivery into the tumors is now consideredto be a potentially effective approach to cancer therapy. On the otherhand, there is a concern that angiogenesis inhibitors inhibit survivalof vascular endothelial cells and induce death of vascular endothelialcells and their interacting vascular mural cells, thereby aggravatingischemia in tumors. Hypoxia in tumors is considered to cause malignanttransformation of cancer cells and facilitate cancer invasion andmetastasis. Also reported is that angiogenesis inhibitors damage bloodvessels in normal tissue and cause severe adverse effects, such ashypertension, lung hemorrhage and renal dysfunction (Non PatentLiterature 4 and 5). Under such circumstances, there has been a demandfor the development of a drug that normalizes the vascular permeabilityin tumors without causing the regression of tumor vessels and withoutaffecting normal blood vessels.

The present inventors have found that lysophosphatidic acid (LPA)administered to subcutaneous tumor-bearing mice activates an LPAreceptor, which mediates the induction of normal web-like networkformation of tumor vessels, the formation of a smooth luminal surface intumor vessels, and the normalization of vascular permeability. Based onthis finding, the present inventors have applied for a patent (PatentLiterature 1).

CITATION LIST Patent Literature

-   Patent Literature 1: WO 2015/152412

Non Patent Literature Non Patent Literature 1:

-   Folkman J, et al.: Isolation of a tumor factor responsible for    angiogenesis. J Exp Med 133: 275-288, 1971

Non Patent Literature 2:

-   Gerber H P, Ferrara N. Pharmacology and pharmacodynamics of    bevacizumab as monotherapy or in combination with cytotoxic therapy    in preclinical studies. Cancer Res 65; 671-680, 2005

Non Patent Literature 3:

-   Jain RK: Normalization of tumor vasculature: An emerging concept in    antiangiogenic therapy. Science 307: 58-62, 2005

Non Patent Literature 4:

-   Ebos JML et al., Accelerated metastasis after short-term treatment    with a potent inhibitor of tumor angiogenesis. Cancer Cell 15:    232-239, 2009

Non Patent Literature 5:

-   Paez-Ribes M et al., Antiangiogenic therapy elicits malignant    progression of tumors to increased local invasion and distant    metastasis. Cancer Cell 12: 220-231, 2009

SUMMARY OF INVENTION Technical Problem

An object of the present disclosure is to find out a substance which iscapable of inducing intratumoral infiltration of immune cells bypromoting connection of discontinuous intratumoral vessels to each otherand promoting intratumoral vein formation without affecting normal bloodvessels. Another object of the present disclosure is to provide a novelapplication of the substance.

Solution to Problem

The present disclosure includes the following to achieve theabove-mentioned object.

(1) A vein-formation promoting agent comprising a phosphatidylcholine asan active ingredient, the agent being capable of promoting vein-likemorphological change of tumor vessels.(2) The vein-formation promoting agent according to the above (1),wherein the agent is capable of enlarging the diameter of tumor vesselsand/or promoting connection of tumor vessels to each other.(3) The vein-formation promoting agent according to the above (1) or(2), wherein the phosphatidylcholine is one kind of phosphatidylcholineor a mixture of two or more kinds of phosphatidylcholines.(4) The vein-formation promoting agent according to any one of the above(1) to (3), wherein the agent is used in combination with cancerimmunotherapy.(5) The vein-formation promoting agent according to the above (4),wherein the cancer immunotherapy is a therapy for reversal ofimmunosuppression and/or immune cell infusion therapy.(6) The vein-formation promoting agent according to the above (5),wherein the therapy for reversal of immunosuppression uses an immunecheckpoint inhibitor, and wherein the immune checkpoint inhibitor is ananti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, a PD-L1blocker or an anti-PD-L1 antibody.(7) A vessel-diameter enlarging agent comprising a phosphatidylcholineas an active ingredient, the agent being capable of enlarging thediameter of tumor vessels.(7-2) The vessel-diameter enlarging agent according to the above (7),wherein the phosphatidylcholine is one kind of phosphatidylcholine or amixture of two or more kinds of phosphatidylcholines.(7-3) The vessel-diameter enlarging agent according to the above (7) or(7-2), wherein the agent is used in combination with cancerimmunotherapy.(7-4) The vessel-diameter enlarging agent according to the above (7-3),wherein the cancer immunotherapy is a therapy for reversal ofimmunosuppression and/or immune cell infusion therapy.(7-5) The vessel-diameter enlarging agent according to the above (7-4),wherein the therapy for reversal of immunosuppression uses an immunecheckpoint inhibitor, and wherein the immune checkpoint inhibitor is ananti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, a PD-L1blocker or an anti-PD-L1 antibody.(8) A blood vessel-connection promoting agent comprising aphosphatidylcholine as an active ingredient, the agent being capable ofpromoting connection of tumor vessels to each other without mediation ofa lysophospholipid receptor.(8-2) The blood vessel-connection promoting agent according to the above(8), wherein the phosphatidylcholine is one kind of phosphatidylcholineor a mixture of two or more kinds of phosphatidylcholines.(8-3) The blood vessel-connection promoting agent according to the above(8) or (8-2), wherein the agent is used in combination with cancerimmunotherapy.(8-4) The blood vessel-connection promoting agent according to the above(8-3), wherein the cancer immunotherapy is a therapy for reversal ofimmunosuppression and/or immune cell infusion therapy.(8-5) The blood vessel-connection promoting agent according to the above(8-4), wherein the therapy for reversal of immunosuppression uses animmune checkpoint inhibitor, and wherein the immune checkpoint inhibitoris an anti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, aPD-L1 blocker or an anti-PD-L1 antibody.(9) A leukocyte-infiltration promoting agent comprising aphosphatidylcholine as an active ingredient, the agent being capable ofpromoting infiltration of leukocytes throughout a tumor region withoutmediation of a lysophospholipid receptor.(9-2) The leukocyte-infiltration promoting agent according to the above(9), wherein the phosphatidylcholine is one kind of phosphatidylcholineor a mixture of two or more kinds of phosphatidylcholines.(9-3) The leukocyte-infiltration promoting agent according to the above(9) or (9-2), wherein the agent is used in combination with cancerimmunotherapy.(9-4) The leukocyte-infiltration promoting agent according to the above(9-3), wherein the cancer immunotherapy is a therapy for reversal ofimmunosuppression and/or immune cell infusion therapy.(9-5) The leukocyte-infiltration promoting agent according to the above(9-4), wherein the therapy for reversal of immunosuppression uses animmune checkpoint inhibitor, and wherein the immune checkpoint inhibitoris an anti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, aPD-L1 blocker or an anti-PD-L1 antibody.(10) The leukocyte-infiltration promoting agent according to the above(9), wherein the leukocytes are CD4-positive cells and/or CD8-positivecells.(11) An antitumor immunostimulatory agent comprising aphosphatidylcholine as an active ingredient, the agent being capable ofpromoting infiltration of leukocytes throughout a tumor region withoutmediation of a lysophospholipid receptor.(11-2) The leukocyte-infiltration promoting agent according to the above(11), wherein the phosphatidylcholine is one kind of phosphatidylcholineor a mixture of two or more kinds of phosphatidylcholines.(11-3) The leukocyte-infiltration promoting agent according to the above(11) or (11-2), wherein the agent is used in combination with cancerimmunotherapy.(11-4) The leukocyte-infiltration promoting agent according to the above(11-3), wherein the cancer immunotherapy is a therapy for reversal ofimmunosuppression and/or immune cell infusion therapy.(11-5) The leukocyte-infiltration promoting agent according to the above(11-4), wherein the therapy for reversal of immunosuppression uses animmune checkpoint inhibitor, and wherein the immune checkpoint inhibitoris an anti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, aPD-L1 blocker or an anti-PD-L1 antibody.(12) The antitumor immunostimulatory agent according to the above (11),wherein the leukocytes are CD4-positive cells and/or CD8-positive cells.

Advantageous Effects of Invention

Phosphatidylcholine, which is an active ingredient of the presentdisclosure, is capable of promoting connection of abnormallydiscontinuous intratumoral vessels to each other and enlarging thediameter of tumor vessels to promote vein formation without affectingnormal blood vessels. It is known that inflammatory cells, such asleukocytes, enter tissues from veins, not from capillaries. Taken thesetogether, phosphatidylcholine has the potential to improve intratumoralblood flow, promote leukocyte infiltration throughout the tumor region,stimulate antitumor immunity in the tumor, and/or inhibit tumor growth.The vein-formation promoting agent, the vessel-diameter enlarging agent,the blood vessel-connection promoting agent, the leukocyte-infiltrationpromoting agent and the antitumor immunostimulatory agent of the presentdisclosure do not destroy tumor vessels but rather diminish the hypoxicarea in a tumor. For this reason, these agents have the advantage of notinducing malignant transformation of cancer cells. Moreover, thevein-formation promoting agent, the vessel-diameter enlarging agent, theblood vessel-connection promoting agent, the leukocyte-infiltrationpromoting agent and/or the antitumor immunostimulatory agent of thepresent disclosure have the advantage of efficiently delivering a drugused in combination with these agents into a tumor.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the tumor growth inhibitory effect of soybeanphosphatidylcholine or Intralipos Injection 20% (trade name, OtsukaPharmaceutical Co., Ltd., containing 1.2 mg of purified yolk lecithin in100 μL) administered for 9 days to tumor-bearing mice generated bysubcutaneous inoculation of a mouse Lewis lung cancer cell line(hereinafter referred to as “LLC cells”).

FIG. 2 shows the structural changes of tumor vessels after 9-dayadministration of soybean phosphatidylcholine or Intralipos Injection20% (trade name, Otsuka Pharmaceutical Co., Ltd.) to tumor-bearing micegenerated by subcutaneous inoculation of LLC cells.

FIG. 3 shows the intratumoral infiltration of CD4-positive cells after9-day administration of soybean phosphatidylcholine or IntraliposInjection 20% (trade name, Otsuka Pharmaceutical Co., Ltd.) totumor-bearing mice generated by subcutaneous inoculation of LLC cells.

FIG. 4 shows the intratumoral infiltration of CD8-positive cells after9-day administration of soybean phosphatidylcholine or IntraliposInjection 20% (trade name, Otsuka Pharmaceutical Co., Ltd.) totumor-bearing mice generated by subcutaneous inoculation of LLC cells.

FIG. 5 shows the structural changes of tumor vessels after 9-dayadministration of soybean phosphatidylcholine or lysophosphatidylcholineto tumor-bearing mice generated by subcutaneous inoculation of LLCcells.

FIG. 6 shows the structural changes of tumor vessels after 7-dayadministration of soybean phosphatidylcholine to tumor-bearing micegenerated by subcutaneous inoculation of colon-26 cells (mouse coloncancer cell line).

FIG. 7 shows the intratumoral infiltration of CD4-positive cells andCD8-positive cells after 7-day administration of soybeanphosphatidylcholine to tumor-bearing mice generated by subcutaneousinoculation of colon-26 cells (mouse colon cancer cell line).

FIG. 8 shows the structural changes of tumor vessels after 7-dayadministration of soybean phosphatidylcholine to tumor-bearing micegenerated by subcutaneous inoculation of B16 cells (mouse melanoma cellline).

FIG. 9 shows the intratumoral infiltration of CD4-positive cells andCD8-positive cells after 7-day administration of soybeanphosphatidylcholine to tumor-bearing mice generated by subcutaneousinoculation of 816 cells (mouse melanoma cell line).

FIG. 10 shows the tumor growth inhibitory effect of combinedadministration of soybean phosphatidylcholine and an anti-PD-1 antibodyto tumor-bearing mice generated by subcutaneous inoculation of LLCcells.

FIG. 11 shows the tumor growth inhibitory effect of combinedadministration of soybean phosphatidylcholine and an anti-PD-1 antibodyto tumor-bearing mice generated by subcutaneous inoculation of colon-26cells.

FIG. 12 shows the intratumoral infiltration of exogenous lymphocytesadministered intravenously on the day following the final day of 7-dayadministration of soybean phosphatidylcholine to tumor-bearing micegenerated by subcutaneous inoculation of colon-26 cells. The exogenouslymphocytes were lymphocytes collected from other mice.

FIG. 13 shows the intratumoral infiltration of exogenous lymphocytesadministered intravenously on the day following the final day of 7-dayadministration of soybean phosphatidylcholine to tumor-bearing micegenerated by subcutaneous inoculation of B16 cells. The exogenouslymphocytes were lymphocytes collected from other mice.

FIG. 14 shows the intratumoral infiltration of exogenous lymphocytesadministered intravenously on the day following the final day of 7-dayadministration of soybean phosphatidylcholine to tumor-bearing micegenerated by subcutaneous inoculation of LLC cells. The exogenouslymphocytes were lymphocytes collected from other mice.

FIG. 15 shows the intratumoral infiltration of CD4-positive cells andCD8-positive cells after 7-day administration of distearoylphosphatidylcholine to tumor-bearing mice generated by subcutaneousinoculation of LLC cells.

FIG. 16 shows the intratumoral hypoxic area after 7-day administrationof soybean phosphatidylcholine or Intralipos Injection 20% (trade name,Otsuka Pharmaceutical Co., Ltd.) to tumor-bearing mice generated bysubcutaneous inoculation of LLC cells.

FIG. 17 shows the intratumoral penetration of doxorubicin administeredon the day following the final day of 7-day administration of soybeanphosphatidylcholine or Intralipos Injection 20% (trade name, OtsukaPharmaceutical Co., Ltd.) to tumor-bearing mice generated bysubcutaneous inoculation of LLC cells.

FIG. 18 shows the tumor growth inhibitory effect of combinedadministration of soybean phosphatidylcholine and 5-FU to tumor-bearingmice generated by subcutaneous inoculation of LLC cells.

FIG. 19 shows the effect of soybean phosphatidylcholine on tubulogenesisof cultured human umbilical vein endothelial cells (HUVECs).

FIG. 20 shows the effect of various phosphatidylcholines ontubulogenesis of cultured human umbilical vein endothelial cells(HUVECs).

DESCRIPTION OF EMBODIMENTS

Phosphatidylcholine is a member of phospholipid and is also calledlecithin. Phospholipids, unlike other lipids, not only serve as anenergy source, but also participate in cellular signal transduction as alipid mediator. Phosphatidylcholine is the most abundant amongphospholipids in a human body and is a major component of cellmembranes. Phosphatidylcholine is also known as a source of theneurotransmitter acetylcholine and is considered to be associated withtransmission of parasympathetic impulses and learning, memory and sleep.In addition, phosphatidylcholine is also known to participate in lipidmetabolism and have hepatoprotective activity. Phosphatidylcholine isphysically characterized by having the features of both water and oil.Phosphatidylcholine is composed of four constituents: phosphoric acid,choline, glycerol and fatty acids. Since phosphoric acid and choline arehydrophilic and glycerol and fatty acids are lipophilic,phosphatidylcholine has emulsifying effect, which makes water and oilinto a smooth mixture. Due to this emulsifying effect,phosphatidylcholine helps intracellular water-soluble substances andintracellular lipophilic substances to mix with each other, and therebycontributes to intracellular absorption of nutrients, excretion ofcellular waste products and other cellular events. In addition, theemulsifying effect leads to activation of lipid metabolism, throughwhich phosphatidylcholine is considered effective for improvement ofhyperlipidemia and for prevention of arteriosclerosis. However, specificeffects of phosphatidylcholine on intratumoral vessels have not beenunderstood at all.

The present inventors administered phosphatidylcholine to tumor-bearingmice generated by subcutaneous inoculation of cancer cells. As a result,tumor growth was inhibited as compared with mice which had not receivedphosphatidylcholine (non-treatment mice). In addition, tumor vessels inthe non-treatment mice were tortuous and irregularly branched, but inthe mice having received phosphatidylcholine, tumor vessels were nolonger discontinuous and were connected to each other to form a web-likenetwork as observed in normal tissue, and some of the tumor vessels hada vein-like morphology with an enlarged diameter. Further, theexamination of immune cell localization in the tumor of the mice havingreceived phosphatidylcholine revealed that a larger number ofCD4-positive cells and CD8-positive cells were present throughout thetumor region including the central region of the tumor as compared withtumor tissue of the non-treatment mice. That is, the present inventorsfound that phosphatidylcholine is capable of promoting infiltration ofimmune cells throughout the tumor region. The above results show thatphosphatidylcholine administration promotes infiltration of CD8-positivecytotoxic T cells and CD4-positive helper T cells into a tumor,resulting in stimulation of antitumor immunity and activation ofcytotoxic T-cell attack on tumor cells, leading to the induction ofantitumor effect.

The present inventors previously found that lysophosphatidic acid (LPA)administered to subcutaneous tumor-bearing mice activates an LPAreceptor, which mediates the induction of normal web-like networkformation of tumor vessels, the formation of a smooth luminal surface intumor vessels, and the normalization of vascular permeability (PatentLiterature 1). Lysophosphatidic acid is enzymatically synthesized fromlysophosphatidylcholine, which is produced by enzymatic decomposition ofphosphatidylcholine. Considering this, there was a possibility that thenewly discovered effects of phosphatidylcholine might be underlain bythe mechanism that the administered phosphatidylcholine undergoes invivo decomposition into lysophosphatidic acid, which activates alysophosphatidic acid receptor and thereby produces the effects.However, the structural improvement of intratumoral vessels and thepromotion of immune cell infiltration after phosphatidylcholineadministration were not observed after lysophosphatidyicholineadministration (see Reference Example 1). Furthermore, in an experimentusing lysophosphatidic acid receptor 4-deficient mice,phosphatidylcholine administration improved the structure ofintratumoral vessels and promoted immune cell infiltration. Therefore,the effects of phosphatidylcholine were proven not to be mediated by alysophosphatidic acid receptor.

In addition, Patent Literature 1 discloses that administration of alysophospholipid receptor-activating substance is effective for inducingtumor vessels, which have been tortuous and irregularly branched beforeadministration, to form a web-like network as observed in normal tissueand for normalizing vascular permeability. However, there is nodescription or suggestion on dilation or vein-like morphological changeof the connected tumor vessels or on immune cell infiltration throughoutthe tumor region. That is, even with the knowledge thatphosphatidylcholine is a precursor of lysophosphatidic acid, theabove-mentioned effects of phosphatidylcholine administration cannot beanticipated from the disclosure of Patent Literature 1.

The present disclosure provides a vein-formation promoting agent whichcomprises a phosphatidylcholine as an active ingredient and is capableof promoting vein-like morphological change of tumor vessels. Thepresent disclosure also provides a vessel-diameter enlarging agent whichcomprises a phosphatidylcholine as an active ingredient and is capableof enlarging the diameter of tumor vessels. The present disclosure alsoprovides a blood vessel-connection promoting agent which comprises aphosphatidylcholine as an active ingredient and is capable of promotingconnection of tumor vessels to each other without mediation of alysophospholipid receptor. The present disclosure also provides aleukocyte-infiltration promoting agent and an antitumorimmunostimulatory agent, each of which comprises a phosphatidylcholineas an active ingredient and is capable of promoting infiltration ofleukocytes throughout a tumor region without mediation of alysophospholipid receptor. Hereinafter, these embodiments of the presentdisclosure are collectively referred to as “the agent of the presentdisclosure.” The phosphatidylcholine used as an active ingredient of theagent of the present disclosure does not encompass a phosphatidylcholinewhich is a component of another agent to be administered in the form ofliposomes or colloidal particles encapsulating or incorporating anothertherapeutic drug.

The phosphatidylcholine (also called lecithin) used as an activeingredient of the agent of the present disclosure is not particularlylimited as long as its structure has ester bonds with fatty acids at theC-1 and C-2 positions of glycerol and an ester bond with phosphocholineat the C-3 position of glycerol. Examples of the phosphatidylcholineinclude yolk lecithin, soybean lecithin, soybean phosphatidylcholine,dioctanoyl phosphatidylcholine, dinonanoyl phosphatidylcholine,didecanoyl phosphatidylcholine, diundecanoyl phosphatidylcholine,dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine,dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine,dipalmitoleoyl phosphatidylcholine, dioleoyl phosphatidylcholine,dilinoleoyl phosphatidylcholine, dieicosapentaenoyl phosphatidylcholine,didocosahexaenoyl phosphatidylcholine, dierucoyl phosphatidylcholine,(1-myristoyl-2-palmitoyl) phosphatidylcholine, (1-palmitoyl-2-myristoyl)phosphatidylcholine, (1-oleoyl-2-palmitoyl) phosphatidylcholine and(1-palmitoyl-2-oleoyl) phosphatidylcholine.

The phosphatidylcholine used in the agent of the present disclosure maybe a phosphatidylcholine extracted from the natural source or aphosphatidylcholine chemically synthesized. Examples of thephosphatidylcholine extracted from the natural source include yolklecithin, soybean lecithin and soybean phosphatidylcholine. Purifiedyolk lecithin and purified soybean lecithin of high quality and highpurity are commercially available, and such commercial products aresuitable for use in the present disclosure. The phosphatidylcholine usedin the present disclosure may contain two or more kinds ofphosphatidylcholines or be composed of one kind of phosphatidylcholine.The phosphatidylcholine extracted from the natural source is usually amixture of several kinds of phosphatidylcholines.

Each fatty acid group of the phosphatidylcholine may be a saturatedfatty acid group or an unsaturated fatty acid group. The number ofcarbon atoms of the fatty acid group is not particularly limited, andmay be 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 15 ormore, 20 or more, or 30 or more. In addition, the number of carbon atomsof the fatty acid group may be 100 or less, 80 or less, 60 or less, 50or less, 40 or less, or 30 or less. The number of double bonds of theunsaturated fatty acid group is not particularly limited, and may be 1or more, 2 or more, 3 or more, 4 or more, or 5 or more. In addition, thenumber of double bonds of the unsaturated fatty acid group may be 6 orless, 5 or less, 4 or less, 3 or less, or 2 or less. The two fatty acidgroups of the phosphatidylcholine may be the same or different from eachother. That is, the two fatty acid groups of the phosphatidylcholine maybe identical saturated fatty acid groups, identical unsaturated fattyacid groups, different saturated fatty acid groups, differentunsaturated fatty acid groups, or a combination of a saturated fattyacid group and an unsaturated fatty acid group.

In the present disclosure, a tumor is a mass of abnormally growing cellsand includes a benign tumor and a malignant tumor. The tumor as a targetof the agent of the present disclosure for promoting vein-likemorphological change of blood vessels and leukocyte infiltration may bea benign tumor or a malignant tumor, and is preferably a malignanttumor. More preferably, the tumor is a solid cancer. In solid cancers,blood vessels are tortuous and irregularly branched, the luminal surfaceis irregular, and vascular permeability is excessively increased. Solidcancers include, but are not limited to, lung cancer, colon cancer,prostate cancer, breast cancer, pancreatic cancer, esophageal cancer,gastric cancer, liver cancer, biliary cancer, spleen cancer, renalcancer, bladder cancer, uterine cancer, ovarian cancer, testicularcancer, thyroid cancer and brain tumor. Solid cancers also include atumor formed from cancerous blood cells.

In the present disclosure, the vein-like morphological change of tumorvessels can be confirmed by, for example, preparing tissue specimens ofa tumor, immunostaining the tissue specimens with an antibody specificto vascular endothelial cells, and measuring the vessel diameter.Specifically, in the case where the vessel diameter is more than 10 μm,the vessel can be judged to have changed into a vein-like form.Preferably, the vessel diameter is 12 μm or more, and is more preferably15 μm or more. In addition, the vein-like morphological change of tumorvessels can also be confirmed by examining the expression of a genespecific to venous cells. Examples of the gene specific to venous cellsinclude EphB4 and COUP-TFII. The gene expression can be examined byknown methods, such as PCR, using the nucleic acid extracted from asample. The vein-like morphological change of tumor vessels may beconfirmed based on a combination of the vessel diameter and the geneexpression specific to venous cells. Specifically, for example, in thecase where the vessel diameter of more than 10 μm and the expression ofEphB4 and COUP-TFII in blood vessel tissue have been confirmed, thetumor vessel can be judged to have changed into a vein-like form.

In the present disclosure, leukocytes include lymphocytes (T cells, Bcells and NK cells), monocytes (macrophages and dendritic cells) andgranulocytes (neutrophils, eosinophils and basophils). The type ofleukocytes whose infiltration throughout a tumor region is promoted bythe agent of the present disclosure is not particularly limited, and theagent of the present disclosure promotes the infiltration of all typesof cells included in the leukocytes as described above. Preferably, theleukocytes are cells serving to stimulate antitumor immunity in tumors(antitumor immune cells). Examples of such cells include cytotoxic Tcells, NK cells, NKT cells, killer cells, macrophages, granulocytes,helper T cells and LAK cells. The leukocytes whose infiltration into thecentral region of a tumor is promoted by the agent of the presentdisclosure are preferably CD4-positive cells and/or CD8-positive cells.The CD4-positive cells are preferably helper T cells, and theCD8-positive cells are preferably cytotoxic T cells. The type andlocation of cells that have infiltrated into a tumor can be identifiedby, for example, preparing tissue specimens of the tumor andimmunostaining the tissue specimens with an antibody against a surfaceantigen specific to the cells of interest.

In some embodiments, the agent of the present disclosure may be in theform of a medicament. That is, the agent of the present disclosure canbe produced in a dosage form by blending phosphatidylcholine as anactive ingredient with a pharmaceutically acceptable carrier or additiveas appropriate according to a known production method for pharmaceuticalpreparations (e.g., the methods described in the Japanese Pharmacopoeia,etc.). Specifically, the agent of the present disclosure may be, forexample, an oral preparation or a parenteral preparation, includingtablets (including sugar-coated tablets, film-coated tablets, sublingualtablets, orally disintegrating tablets, and buccal tablets), pills,powders, granules, capsules (including soft capsules and microcapsules),troches, syrups, liquids, emulsions, suspensions, controlled-releasepreparations (e.g., fast-release preparations, sustained releasepreparations, sustained release microcapsules, etc.), aerosols, films(e.g., orally disintegrating films, oral mucosal adhesive films, etc.),injections (e.g., subcutaneous injections, intravenous injections,intramuscular injections, intraperitoneal injections, etc.), intravenousinfusions, transdermal preparations, ointments, lotions, patches,suppositories (e.g., rectal suppositories, vaginal suppositories, etc.),pellets, transnasal preparations, transpulmonary preparations(inhalants), and eye drops. The amount of the carrier or the additive tobe added is determined as appropriate based on the range of amountconventionally used in the pharmaceutical field. The carrier or theadditive that can be used is not particularly limited, and examplesinclude various carriers such as water, physiological saline, otheraqueous solvents, and aqueous or oily bases; and various additives suchas fillers, binders, pH adjusters, disintegrants, absorption enhancers,lubricants, colorants, corrigents and fragrances.

Examples of the additive that can be blended into tablets, capsules andthe like include binders such as gelatin, cornstarch, tragacanth and gumarabic; fillers such as crystalline cellulose; bulking agents such ascornstarch, gelatin and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose and saccharin; and flavorssuch as peppermint, Gaultheria adenothrix oil and cherry. In the casewhere the unit dosage form is a capsule, a liquid carrier such as fatsand oils can be further contained in addition to the above-mentionedingredients. A sterile composition for injection can be preparedaccording to the usual procedure for pharmaceutical formulation, forexample, by dissolving or suspending an active ingredient in a solventsuch as water for injection and a natural vegetable oil. As an aqueousliquid for injection, for example, physiological saline, an isotonicsolution containing glucose and an auxiliary substance (e.g.,D-sorbitol, D-mannitol, sodium chloride, etc.), or the like can be used,optionally together with a suitable solubilizer such as alcohols (e.g.,ethanol etc.), polyalcohols (e.g., propylene glycol, polyethyleneglycol, etc.) and nonionic surfactants (e.g., polysorbate 80™, HCO-50,etc.). As an oily liquid, for example, sesame oil, soybean oil or thelike can be used, optionally together with a solubilizer such as benzylbenzoate and benzyl alcohol. Further, a buffering agent (e.g., phosphatebuffer, sodium acetate buffer, etc.), a soothing agent (e.g.,benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer(e.g., human serum albumin, polyethylene glycol, etc.), a preservative(e.g., benzyl alcohol, phenol, etc.), an antioxidant and/or the like mayalso be added.

In some embodiments, the agent of the present disclosure may be in theform of a lipid emulsion containing phosphatidylcholine in a vegetableoil. Examples of the vegetable oil include soybean oil, corn oil,coconut oil, safflower oil, perilla oil, olive oil, castor oil andcottonseed oil. Preferred is soybean oil. For example, “IntraliposInjection 10% (trade name)” and “Intralipos Injection 20% (trade name)”(both are manufactured by Otsuka Pharmaceutical Co., Ltd.), which arelisted in the National Health Insurance (NHI) Drug Price List aspharmaceutical products for nutritional support in the pre- orpost-operative or other appropriate periods, contain purified soybeanoil as an active ingredient and purified yolk lecithin (1.2 g/100 mL) asan additive. Therefore, “Intralipos Injection 10% (trade name)” and“Intralipos Injection 20% (trade name)” are suitable for embodiments ofthe agent of the present disclosure.

Phosphatidylcholine is a substance present in a living body and has beenpractically used as an active ingredient or an additive inpharmaceutical products for administration to humans. Therefore,phosphatidylcholine is less toxic and safely administrable to humans andother mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats,dogs, monkeys, etc.).

The amount of an active ingredient contained in pharmaceuticalpreparations is determined as appropriate for the dosage form, theadministration method, the carrier and the like. The amount ofphosphatidylcholine can be usually 0.01 to 100% (w/w), preferably 0.1 to95% (w/w) relative to the total weight of the pharmaceuticalpreparation.

The dose of phosphatidylcholine may vary depending on the subject, thesymptoms, the administration route and the like, but in general, thedaily oral dose for a human weighing about 60 kg is, for example, about0.01 to 1000 mg, preferably about 0.1 to 100 mg, and more preferablyabout 0.5 to 500 mg. The single dose for parenteral administration mayalso vary depending on patient's condition, the symptoms, theadministration method and the like, but for example in the case ofinjection preparations, the single intravenous dose is usually, forexample, about 0.01 to 100 mg, preferably about 0.01 to 50 mg, and morepreferably about 0.01 to 20 mg per kg of body weight. The total dailydose may be given as a single dose or in divided doses.

The agent of the present disclosure may be used in combination withcancer immunotherapy. The agent of the present disclosure, when used incombination with cancer immunotherapy, can stimulate antitumor immunityand increase tumor cytotoxicity. The phrase “the agent of the presentdisclosure is used in combination with cancer immunotherapy” means thatthe agent of the present disclosure is used for administration to acancer patient receiving cancer immunotherapy or that the agent of thepresent disclosure is used in combination (concomitantly) with a drugfor cancer immunotherapy. When the agent of the present disclosure isused in combination with cancer immunotherapy, the dose of the drug forcancer immunotherapy can be reduced, which may lead to reduced sideeffects. Moreover, the reduction in the dose of the drug for cancerimmunotherapy meets social needs including healthcare cost reduction. Asused herein, the terms “used in combination” and “used concomitantly”have the same meaning.

Examples of the cancer immunotherapy include cancer vaccine therapy,immune cell infusion therapy, a therapy for reversal ofimmunosuppression and a therapy for inducing the depletion of regulatoryT cells. In some embodiments, the cancer immunotherapy may be a therapyfor reversal of immunosuppression or immune cell infusion therapy. Theimmune checkpoint inhibitor used in the therapy for reversal ofimmunosuppression is, for example, an anti-CTLA-4 antibody, a PD-1blocker, an anti-PD-1 antibody, a PD-L1 blocker, an anti-PD-L1 antibody,or the like. Examples of the immune cell infusion therapy includechimeric antigen receptor-modified T-cell therapy. In the case where theagent of the present disclosure is administered after depletion ofregulatory T cells, the same effect as produced by a combined use of theagent of the present disclosure and an immune checkpoint inhibitor isexpected. This is because regulatory T cells play a role inimmunological tolerance. Examples of the drug that induces the depletionof regulatory T cells include alkylating agents, an IL-2-diphtheriatoxin fusion protein, an anti-CD25 antibody, an anti-KIR antibody, anIDO inhibitor and a BRAF inhibitor.

Examples of the drug for cancer immunotherapy include Picibanil,Krestin, sizofiran, lentinan, ubenimex, interferons, interleukins,macrophage colony-stimulating factor, granulocyte colony-stimulatingfactor, erythropoietin, lymphotoxins, BCG vaccine, Corynebacteriumparvum, levamisole, polysaccharide K, procodazole, ipilimumab,nivolumab, ramucirumab, ofatumumab, panitumumab, pembrolizumab,obinutuzumab, trastuzumab emtansine, tocilizumab, bevacizumab,trastuzumab, siltuximab, cetuximab, infliximab, rituximab, metformin andaflibercept.

When the agent of the present disclosure is used in combination withcancer vaccine, efficient infiltration of cancer vaccine-stimulated Tcells into a tumor can be achieved. In addition, the agent of thepresent disclosure can enhance the efficacy of immune cell infusiontherapy, which uses immune cells such as T cells from a patient or anon-patient.

Since the combined use of the agent of the present disclosure and cancerimmunotherapy can enhance cancer immunotherapy and increase tumorcytotoxicity as described above, the agent of the present disclosureaccording to an embodiment where the agent is used in combination withcancer immunotherapy can be called a cancer immunotherapy-enhancingagent.

The agent of the present disclosure can be used in combination with ananticancer drug other than those described above. Due to the antitumorimmunostimulatory effect of the agent of the present disclosure, theoriginal anticancer effect of the anticancer drug can be enhanced. Thus,the dose of the anticancer drug can be reduced, which may lead toreduced side effects. Moreover, the reduction in the dose of theanticancer drug meets social needs including healthcare cost reduction.

The anticancer drug is not particularly limited and is preferably, forexample, a chemotherapeutic drug, an immunotherapeutic drug or a hormonetherapy drug. These anticancer drugs may be in the form of a liposomalformulation. These anticancer drugs may be in the form of a nucleic acidformulation or an antibody formulation.

The chemotherapeutic drug is not particularly limited and examplesinclude alkylating agents such as nitrogen mustard, nitrogen mustardN-oxide hydrochloride, chlorambucil, cyclophosphamide, ifosfamide,thiotepa, carboquone, improsulfan tosilate, busulfan, nimustinehydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine,estramustine phosphate sodium, triethylenemelamine, carmustine,lomustine, streptozocin, pipobroman, ethoglucid, carboplatin, cisplatin,miboplatin, nedaplatin, oxaliplatin, altretamine, ambamustine,dibrospidium chloride, fotemustine, prednimustine, pumitepa, Ribomustin,temozolomide, treosulfan, trofosfamide, zinostatin stimalamer,adozelesin, cystemustine and bizelesin; antimetabolites such asmercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate,pemetrexed, enocitabine, cytarabine, cytarabine ocfosfate, ancitabinehydrochloride, 5-FU and its derivatives (e.g., fluorouracil, tegafur,UFT, doxifluridine, carmofur, galocitabine, emitefur, capecitabine,etc.), aminopterin, nelzarabine, leucovorin calcium, Tabloid, butocin,calcium folinate, calcium levofolinate, cladribine, emitefur,fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim,idoxuridine, mitoguazone, tiazofurin, ambamustine and bendamustine;anticancer antibiotics such as actinomycin D, actinomycin C, mitomycinC, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate,peplomycin sulfate, daunorubicin hydrochloride, doxorubicinhydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride,epirubicin hydrochloride, neocarzinostatin, mithramycin, sarkomycin,carzinophilin, mitotane, zorubicin hydrochloride, mitoxantronehydrochloride and idarubicin hydrochloride; and plant-derived anticancerdrugs such as etoposide, etoposide phosphate, vinblastine sulfate,vincristine sulfate, vindesine sulfate, teniposide, paclitaxel,docetaxel, vinorelbine, irinotecan, and irinotecan hydrochloride.

The immunotherapeutic drug is not particularly limited and examplesinclude Picibanil, Krestin, sizofiran, lentinan, ubenimex, interferons,intexleukins, macrophage colony-stimulating factor, granulocytecolony-stimulating factor, erythropoietin, lymphotoxins, BCG vaccine,Corynebacterium parvum, levamisole, polysaccharide K, procodazole,ipilimumab, nivolumab, ramucirumab, ofatumumab, panitumumab,pembrolizumab, obinutuzumab, trastuzumab emtansine, tocilizumab,bevacizumab, trastuzumab, siltuximab, cetuximab, infliximab andrituximab.

The hormone therapy drug is not particularly limited and examplesinclude fosfestrol, diethylstilbestrol, chlorotrianisene,medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate,cyproterone acetate, danazol, allylestrenol, gestrinone, mepartricin,raloxifene, ormeloxifene, levormeloxifene, antiestrogens (e.g.,tamoxifen citrate, toremifene citrate, etc.), birth-control pills,mepitiostane, testololactone, aminoglutethimide, LH-RH agonists (e.g.,goserelin acetate, buserelin, leuprorelin, etc.), droloxifene,epitiostanol, ethinylestradiol sulfonate, aromatase inhibitors (e.g.,fadrozole hydrochloride, anastrozole, letrozole, exemestane, vorozole,formestane, etc.), antiandrogens (e.g., flutamide, bicalutamide,nilutamide, etc.), 5α-reductase inhibitors (e.g., finasteride,epristeride, etc.), corticosteroids (e.g., dexamethasone, prednisolone,betamethasone, triamcinolone, etc.) and androgen synthesis inhibitors(e.g., abiraterone, etc.).

In the case where the agent of the present disclosure is used incombination (concomitantly) with the immune checkpoint inhibitor oranother anticancer drug, they may be simultaneously administered to asubject or separately administered thereto at some interval. The term“used in combination (concomitantly)” herein means that the period oftreatment with one drug overlaps with the period(s) of treatment withanother or other drugs, and the two or more drugs are not necessarilyrequired to be simultaneously administered. The dose of the immunecheckpoint inhibitor or another anticancer drug can be determined basedon the clinical dose of each drug and is appropriately selecteddepending on the subject, the age and body weight of the subject, thesymptoms, the administration time, the dosage form, the administrationmethod, the combination of the drugs, etc.

The present disclosure further includes the following:

a method for promoting vein-like morphological change of tumor vessels,comprising administering a phosphatidylcholine to a mammal;

a phosphatidylcholine for use in promoting vein-like morphologicalchange of tumor vessels;

use of a phosphatidylcholine for production of a vein-formationpromoting agent capable of promoting vein-like morphological change oftumor vessels;

a method for enlarging the diameter of tumor vessels, comprisingadministering a phosphatidylcholine to a mammal;

a phosphatidylcholine for use in enlarging the diameter of tumorvessels;

use of a phosphatidylcholine for production of a vessel-diameterenlarging agent capable of enlarging the diameter of tumor vessels;

a method for promoting connection of tumor vessels to each other withoutmediation of a lysophospholipid receptor, comprising administering aphosphatidylcholine to a mammal;

a phosphatidylcholine for use in promoting connection of tumor vesselsto each other without mediation of a lysophospholipid receptor;

use of a phosphatidylcholine for production of a blood vessel-connectionpromoting agent capable of promoting connection of tumor vessels to eachother without mediation of a lysophospholipid receptor;

a method for promoting infiltration of leukocytes throughout a tumorregion without mediation of a lysophospholipid receptor, comprisingadministering a phosphatidylcholine to a mammal;

a phosphatidylcholine for use in promoting infiltration of leukocytesthroughout a tumor region without mediation of a lysophospholipidreceptor;

use of a phosphatidylcholine for production of a leukocyte-infiltrationpromoting agent capable of promoting infiltration of leukocytesthroughout a tumor region without mediation of a lysophospholipidreceptor;

a method for stimulating antitumor immunity, comprising administering aphosphatidylcholine to a mammal;

a phosphatidylcholine for use in stimulating antitumor immunity withoutmediation of a lysophospholipid receptor;

use of a phosphatidylcholine for production of an antitumorimmunostimulatory agent capable of stimulating antitumor immunitywithout mediation of a lysophospholipid receptor;

a method for enhancing cancer immunotherapy, comprising administering aphosphatidylcholine to a mammal;

a phosphatidylcholine for use in enhancing cancer immunotherapy withoutmediation of a lysophospholipid receptor;

use of a phosphatidylcholine for production of a cancerimmunotherapy-enhancing agent capable of enhancing cancer immunotherapywithout mediation of a lysophospholipid receptor;

a cancer therapeutic agent comprising a phosphatidylcholine as an activeingredient;

a method for treating cancer, comprising administering aphosphatidylcholine to a mammal;

a phosphatidylcholine for use in cancer therapy; and

use of a phosphatidylcholine for production of a cancer therapeuticagent.

EXAMPLES

Hereinafter, some examples of the present disclosure will be explainedin detail, but the present disclosure is not limited thereto.

Example 1: Effects of Phosphatidylcholine Administration on Tumor (LungCancer)

In order to investigate the effects of phosphatidylcholineadministration on tumor, phosphatidylcholine (hereinafter referred to as“PC”) or a PC-containing purified soybean oil emulsion was administeredto tumor-bearing mice generated by subcutaneous inoculation of a mousecancer cell line, and the antitumor effect, tumor vasculature andintratumoral lymphocyte infiltration were examined.

(1) Experimental Method (1-1) Cells and Animals Used

The mouse cancer cells used were LLC cells (Lewis lung cancer cellline). LLC cells (1×10⁶ cells in 100 μL of PBS per animal) weresubcutaneously injected into C57BL/6NCrSlc mice aged 8 weeks (females,SLC, Inc.).

(1-2) Substances Administered

The PC used was soybean PC (L-α-phosphatidylcholine (95%) (Soy), AvantiPOLAR LIPIDS). The soybean PC was dissolved at a concentration of 25 mMin 50% ethanol, and the solution was stored at −30′C. Just beforeadministration, the soybean PC solution was diluted to a concentrationfor dosing at 3 mg/kg in 100 μL of PBS. The diluted soybean PC solutionwas administered in a volume of 100 μL per administration via the mousetail vein. The PC-containing purified soybean oil emulsion used wasIntralipos Injection 20% (trade name, Otsuka Pharmaceutical Co., Ltd.).The PC-containing purified soybean oil emulsion was administered in avolume of 100 μL (containing 1.2 mg of purified yolk lecithin) peradministration via the mouse tail vein.

(1-3) Grouping and Administration Schedule

The mice were assigned to 3 groups: a soybean PC group, an Intraliposgroup and a control group (non-administration group) (4 animals pergroup). For 9 consecutive days from day 5 to day 13 post-cancer cellinoculation, 100 μL of Intralipos or 3 mg/kg (100 μL) of the soybean PCwas administered once daily via the mouse tail vein with a 27G syringe.On the day following the final administration (day 14 post-inoculation),the tumors were harvested from the mice.

(1-4) Measurement of Tumor Volume

Tumor volume was measured on day 5, day 7, day 10 and day 14post-inoculation. The tumor volume was calculated by the followingformula: length×width×height×0.5.

(1-5) Preparation of Tumor Tissue Specimens

The harvested tumors were immersed in 4% paraformaldehyde (PFA)/PBS andincubated with agitation at 4° C. overnight for fixation. Afterfixation, the tumors were washed with cold PBS (4° C.) for 6 hours,during which PBS was replaced with a fresh one every 30 minutes. Thetumors were immersed in 15% sucrose/PBS and incubated with agitation at4° C. for 3 hours. The tumors were then immersed in 30% sucrose/PBS andincubated with agitation at 4° C. for 3 hours. The tumors were embeddedin O.C.T. compound (Tissue-Tek) and frozen at −800° C. for 3 days orlonger.

(1-6) Observation of Tumor Vasculature The tumors embedded in O.C.T.compound were sectioned at a thickness of 40 μm with a cryostat (Leica).The sections were placed on glass slides and air-dried for about 2 hourswith a dryer. The sections were encircled with a liquid blocker. Theglass slides were placed in a slide staining tray and washed with PBS atroom temperature for 10 minutes to remove the O.C.T. compound. Thesections were post-fixed in 4% PFA/PBS at room temperature for 10minutes and washed with PBS at room temperature for 10 minutes. Ablocking solution (5% normal goat serum, 1% BSA and 2% skim milk in PBS)was applied dropwise to the sections, and blocking was performed at roomtemperature for 20 minutes. As a primary antibody, Purified HamsterAnti-PECAM-1 Antibody (Millipore: MAB1398Z), which is an anti-mouse CD31antibody, was used. This primary antibody was diluted 200-fold in theblocking solution and applied dropwise to the sections. The sectionswere incubated at 4′=C overnight. The sections were washed 5 times withPBS containing Tween 20 (PBST) for 10 minutes each time and further withPBS for 10 minutes. As a secondary antibody, Alexa Fluor 488 GoatAnti-Hamster IgG (Jackson ImmunoResearch Laboratories) was used. Thissecondary antibody was diluted 400-fold in the blocking solution andapplied dropwise to the sections. The sections were incubated in alight-shielding condition for 2 hours. The sections were washed 5 timeswith PBST for 10 minutes each time. Several drops of Vectashield (VectorLaboratories Inc.) were applied to the sections, and glass coverslipswere placed over the sections. The coverslipped sections were observedand photographed under a confocal laser microscope (Leica).

(1-7) Examination of Intratumoral Lymphocyte Infiltration

The tumors embedded in O.C.T. compound were sectioned at a thickness of20 μm with a cryostat (Leica). Post-fixation and blocking were performedaccording to the same procedure as described above. As a primaryantibody, Purified Rat Anti-Mouse CD8 (BioLegend, Inc.: 100801) wasused. The primary antibody was diluted 200-fold in the blocking solutionand applied dropwise to the sections. The sections were incubated at 4°C. overnight. As a secondary antibody, Goat anti-Rat IgG (H+L) SecondaryAntibody, Alexa Fluor 488 conjugate (Thermo Fisher Scientific Inc.:A11006) was used. The secondary antibody was diluted 400-fold in theblocking solution and applied dropwise to the sections. The sectionswere incubated in a light-shielding condition for 2 hours. The sectionswere washed with PBST overnight. As third antibodies, Anti-Mouse CD4 PE(eBioscience: 12-0042-83) and APC-conjugated Rat Anti-Mouse CD31 (BDPharmingen: 551262) were used. The third antibodies were separatelydiluted 400-fold in the blocking solution and applied dropwise to thesections. The sections were incubated in a light-shielding condition for2 hours. The sections were washed with PBST overnight. A mounting medium(DAKO mounting medium, DAKO) was applied dropwise to the sections, andglass coverslips were placed over the sections. The coverslippedsections were photographed under a confocal laser microscope (Leica) at400-fold magnification to obtain confocal images at an optical sectionthickness of 10 μm. The central region and the edge region of the tumorwere separately photographed, and the difference in lymphocyteinfiltration between these regions was examined. The number ofCD4-positive cells and the number of CD8-positive cells in each imagewere separately counted, and the respective lymphocyte count per unitarea was calculated.

(2) Results (2-1) Tumor Volume

The results are shown in FIG. 1. As is clear from FIG. 1, the soybean PCgroup and the Intralipos group showed tumor growth inhibition ascompared with the control group.

(2-2) Tumor Vasculature

The results are shown in FIG. 2. Vascular endothelial cells are stainedin green fluorescence and visualized in white in each image. In thecontrol group, poorly connected blood vessels were observed. Incontrast, well connected blood vessels were observed in the soybean PCgroup and the Intralipos group. In addition, the diameters of most ofthe blood vessels were enlarged to a vein-like morphology with adiameter of more than 10 μm.

(2-3) Intratumoral Lymphocyte Infiltration

The results of CD4-positive cells are shown in FIG. 3, and the resultsof CD8-positive cells are shown in FIG. 4. As is clear from FIGS. 3 and4, the soybean PC group and the Intralipos group showed diffuseinfiltration of both CD4-positive cells and CD8-positive cells into thetumor, evidently indicating the enhancement of antitumor immunity, asdistinct from the control group. The numbers of CD4-positive cells inthe edge region of the tumor in the soybean PC group and in theIntralipos group were 1.5-fold and 1.2-fold higher than that in thecontrol group, respectively. The numbers of CD4-positive cells in thecentral region of the tumor in the soybean PC group and in theIntralipos group were 3-fold and 4-fold higher than that in the controlgroup, respectively. The numbers of CD8-positive cells in the edgeregion of the tumor in the soybean PC group and in the Intralipos groupwere 2-fold or more higher than that in the control group. Similarly,the numbers of CD8-positive cells in the central region of the tumor inthe soybean PC group and in the Intralipos group were 2-fold or morehigher than that in the control group.

(2-4) Conclusion

As is known in the art, inflammatory cells, such as lymphocytes, migrateacross venules, which are blood vessels larger than capillaries, toenter tissues, as previously described. The results of Example 1 showthat PC helps the formation of a connected vascular network, therebyimproving intratumoral blood flow as well as induces vein-likemorphological change of tumor vessels, and via these effects, promotesinfiltration of lymphocytes into tumor tissue, resulting in tumor growthinhibition.

Reference Example 1: Comparison with LysophosphatidylcholineAdministration

An experiment was performed to examine whether administration oflysophosphatidylcholine (hereinafter referred to as “LPC”), which is adecomposition product of PC, would induce vein-like morphological changeof intratumoral vasculature as with the administration of PC.

(1) Experimental Method

The same experiment as described in Example 1 was performed except thatLPC (manufactured by Avanti POLAR LIPIDS;1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine) and soybean PC (the sameas in Example 1) were administered. An LPC solution was prepared at aconcentration for dosing at 3 mg/kg in 100 μL in the same manner as forthe soybean PC solution in Example 1. The LPC solution was administeredin a volume of 100 μL per administration via the mouse tail vein.

(2) Results

The results are shown in FIG. 5. Unlike the soybean PC group, the LPCgroup did not show any structural change of intratumoral vessels. Theseresults revealed that the structural change of intratumoral vessels isinduced by PC itself, not after PC is decomposed into LPC. That is, themechanism to stimulate antitumor immunity in the present disclosure isdifferent from that in the prior invention, which mediates alysophospholipid receptor.

Phosphatidylcholine has been proven safe for use in administration to aliving body. The above findings show that phosphatidylcholine is capableof promoting infiltration of immune cells throughout a tumor region,thus enhancing tumor cell killing by the immune cells, such as cytotoxicT cells. Also shown is that intratumoral infiltration of CD4-positivecells contributes to creating a condition allowing antigen presentationof molecules expressed in tumor cells. Moreover, phosphatidylcholinecauses no damage to blood vessels in normal tissue and therefore has avery low risk of side effects. Phosphatidylcholine seems to exert suchfunctions regardless of the type of cancer and is therefore potentiallyapplicable to any type of cancer. Particularly, phosphatidylcholine isexpected to exert remarkable effect on a type of cancer characterized bylow blood flow (pancreatic cancer etc.).

Example 2: Effects of Phosphatidylcholine Administration on Colon Cancer(1) Experimental Method (1-1) Cells and Animals Used

The mouse cancer cells used were colon-26 cells (mouse colon cancer cellline). Colon-26 cells (1×10⁴ cells in 100 μL of PBS per animal) weresubcutaneously injected into Balb/c mice aged 8 weeks (females, SLC,Inc.).

(1-2) Substance Administered

The same soybean PC as in Example 1 was used. In the same manner as inExample 1, just before administration, the soybean PC solution wasdiluted to a concentration for dosing at 3 mg/kg in 100 μL of PBS. Thediluted soybean PC solution was administered in a volume of 100 μL peradministration via the mouse tail vein.

(1-3) Grouping and Administration Schedule

The mice were assigned to 2 groups: a soybean PC group and a controlgroup (vehicle administration group) (3 animals per group). For 7consecutive days from day 7 to day 13 post-cancer cell inoculation, 3mg/kg (100 μL) of the soybean PC was administered once daily via themouse tail vein. On the day following the final administration (day 14post-inoculation), the tumors were harvested from the mice.

(1-4) Preparation of Tumor Tissue Specimens, Observation of TumorVasculature, and Examination of Intratumoral Lymphocyte Infiltration

In the same manner as in Example 1, tumor tissue specimens wereprepared, tumor vasculature was observed, and intratumoral lymphocyteinfiltration was examined.

(2) Results (2-1) Tumor Vasculature

The results are shown in FIG. 6. Similarly to the results of Example 1,the soybean PC group showed well connected blood vessels having avein-like morphology with a large diameter.

(2-2) Intratumoral Lymphocyte Infiltration

The results are shown in FIG. 7. The left panels show the results ofCD4-positive cells, and the right panels show the results ofCD8-positive cells. Similarly to the results of Example 1, the soybeanPC group showed infiltration of both CD4-positive cells and CD8-positivecells into the tumor.

Example 3: Effects of Phosphatidylcholine Administration on Melanoma (1)Experimental Method (1-1) Cells and Animals Used

The mouse cancer cells used were B16 cells (mouse melanoma cell line).B16 cells (1×10⁶ cells in 100 μL of PBS per animal) were subcutaneouslyinjected into C57BL/6NCrSlc mice aged 8 weeks (females, SLC, Inc.).

(1-2) Substance Administered

The same soybean PC as in Example 1 was used. In the same manner as inExample 1, just before administration, the soybean PC solution wasdiluted to a concentration for dosing at 3 mg/kg in 100 μL of PBS. Thediluted soybean PC solution was administered in a volume of 100 μL peradministration via the mouse tail vein.

(1-3) Grouping and Administration Schedule

The mice were assigned to 2 groups: a soybean PC group and a controlgroup (vehicle administration group) (3 animals per group). For 7consecutive days from day 5 to day 11 post-cancer cell inoculation, 3mg/kg (100 μL) of the soybean PC was administered once daily via themouse tail vein. On the day following the final administration (day 12post-inoculation), the tumors were harvested from the mice.

(1-4) Preparation of Tumor Tissue Specimens, Observation of TumorVasculature, and Examination of Intratumoral Lymphocyte Infiltration

In the same manner as in Example 1, tumor tissue specimens wereprepared, tumor vasculature was observed, and intratumoral lymphocyteinfiltration was examined.

(2) Results (2-1) Tumor Vasculature

The results are shown in FIG. 8. Similarly to the results of Examples 1and 2, the soybean PC group showed well connected blood vessels having avein-like morphology with a large diameter.

(2-2) Intratumoral Lymphocyte Infiltration

The results are shown in FIG. 9. The left panels show the results ofCD4-positive cells, and the right panels show the results ofCD8-positive cells. Similarly to the results of Examples 1 and 2, thesoybean PC group showed infiltration of both CD4-positive cells andCD8-positive cells into the tumor.

The results of Examples 2 and 3 demonstrate that PC exhibits effectssimilar to those observed in Example 1 despite the difference in thetype of cancer and the strain of mice. In conclusion, the effects of PCon intratumoral vessels and lymphocyte infiltration can be exertedregardless of the type of cancer.

Example 4: Combined Effect of Phosphatidylcholine and Immune CheckpointInhibitor on Lewis Lung Cancer (1) Experimental Method (1-1) Cells andAnimals Used

The cells used were LLC cells (Lewis lung cancer cell line). LLC cells(1×10⁴ cells in 100 μL of PBS per animal) were subcutaneously injectedinto C57BL/6NCrSlc mice aged 8 weeks (females, SLC, Inc.).

(1-2) Substances Administered

The same soybean PC as in Example 1 was used. In the same manner as inExample 1, just before administration, the soybean PC solution wasdiluted to a concentration for dosing at 3 mg/kg in 100 μL of PBS. Thediluted soybean PC solution was administered in a volume of 100 μL peradministration via the mouse tail vein. An anti-PD-1 antibody (Bio Xcell) was used as an immune checkpoint inhibitor.

(1-3) Grouping and Administration Schedule

The mice were assigned to 4 groups: a soybean PC group, an anti-PD-1antibody group, a soybean PC plus anti-PD-1 antibody group, and acontrol group (vehicle administration group) (3 animals per group). For13 consecutive days from day 7 to day 20 post-cancer cell inoculation, 3mg/kg (100 μL) of the soybean PC was administered once daily via themouse tail vein. The anti-PD-1 antibody was intraperitoneallyadministered at a dose of 200 μg/mouse on day 7, day 9, day 11, day 14,day 16 and day 18 post-cancer cell inoculation.

(1-4) Measurement of Tumor Volume

Tumor volume was measured on day 7, day 14 and day 21 post-cancer cellinoculation. The tumor volume was calculated by the following formula:length×width×height×0.5.

(2) Results

The measurement results of the tumor volume are shown in FIG. 10.According to previous reports, anti-PD-1 antibodies alone are noteffective against Lewis lung cancer. In accord with the previousreports, the anti-PD-1 antibody group in the present experiment did notshow tumor growth inhibition. On the other hand, the soybean PC groupshowed tumor growth inhibition, and the soybean PC plus anti-PD-1antibody group showed remarkable tumor growth inhibition, which wasconsidered due to a synergistic effect by the combined use of thesesubstances.

Example 5: Combined Effect of Phosphatidylcholine and Immune CheckpointInhibitor on Colon Cancer (1) Experimental Method (1-1) Cells andAnimals Used

The cells used were colon-26 cells (mouse colon cancer cell line).Colon-26 cells (1×10⁶ cells in 100 μL of PBS per animal) weresubcutaneously injected into Balb/c mice aged 8 weeks (females, SLC,Inc.).

(1-2) Substances Administered

The same soybean PC as in Example 1 was used. In the same manner as inExample 1, just before administration, the soybean PC solution wasdiluted to a concentration for dosing at 3 mg/kg in 100 μL of PBS. Thediluted soybean PC solution was administered in a volume of 100 μL peradministration via the mouse tail vein. An anti-PD-1 antibody (Bio Xcell) was used as an immune checkpoint inhibitor.

(1-3) Grouping and Administration Schedule

The mice were assigned to 4 groups: a soybean PC group, an anti-PD-1antibody group, a soybean PC plus anti-PD-1 antibody group, and acontrol group (vehicle administration group) (3 animals per group). For13 consecutive days from day 7 to day 20 post-cancer cell inoculation, 3mg/kg (100 μL) of the soybean PC was administered once daily via themouse tail vein. The anti-PD-1 antibody was intraperitoneallyadministered at a dose of 200 μg/mouse on day 7, day 9, day 11, day 14,day 16 and day 18 post-cancer cell inoculation.

(1-4) Measurement of Tumor Volume

Tumor volume was measured on day 7, day 14 and day 21 post-cancer cellinoculation. The tumor volume was calculated by the following formula:length×width×height×0.5.

(2) Results

The measurement results of the tumor volume are shown in FIG. 11.Similarly to the results of Example 4, the soybean PC group and theanti-PD-1 antibody group showed tumor growth inhibition, and the soybeanPC plus anti-PD-1 antibody group showed remarkable tumor growthinhibition.

The results of Examples 4 and 5 demonstrate that PC induces intratumoralinfiltration of lymphocytes and the immune checkpoint inhibitorstimulates the lymphocytes, thereby remarkably enhancing the antitumoreffect of the lymphocytes regardless of the type of cancer.

Example 6: Combined Effect of Phosphatidylcholine and Immune CellInfusion Therapy on Colon Cancer

Currently available clinical tumor immunotherapies include immune cellinfusion therapy besides immune checkpoint inhibitor therapy. The immunecell infusion therapy involves in vitro proliferation or stimulation ofimmune cells, such as lymphocytes, collected from a cancer patient, andsubsequent administration of the immune cells back to the cancer patientwith the aim of enhancing antitumor immunity. However, sinceintratumoral infiltration of lymphocytes is limited in cancer patients,the requirement to efficiently perform immune cell infusion therapy isto precondition intratumoral vessels to facilitate intratumorallymphocyte infiltration. In this study, an experiment was performed toexamine whether intratumoral lymphocyte infiltration would be promotedin cancer-bearing mice having received PC administration followed byintravenous administration of lymphocytes.

(1) Experimental Method (1-1) Cells and Animals Used

The cells used were colon-26 cells (mouse colon cancer cell line).Colon-26 cells (1×10⁶ cells in 100 μL of PBS per animal) weresubcutaneously injected into Balb/c mice aged 8 weeks (females, SLC,Inc.).

(1-2) Substance Administered

The same soybean PC as in Example 1 was used. In the same manner as inExample 1, just before administration, the soybean PC solution wasdiluted to a concentration for dosing at 3 mg/kg in 100 μL of PBS. Thediluted soybean PC solution was administered in a volume of 100 μL peradministration via the mouse tail vein.

(1-3) Preparation of Lymphocytes

Lymphocytes were harvested from the spleens of transgenic mice(C57BL/6-Tg (CAG-EGFP), SLC, Inc.), which express green fluorescence incells in most of the tissues throughout the body, and a lymphocytesuspension was prepared.

(1-4) Grouping and Administration Schedule

The mice were assigned to 2 groups: a soybean PC group and a controlgroup (vehicle administration group) (3 animals per group). For 7consecutive days from day 7 to day 13 post-cancer cell inoculation, 3mg/kg (100 μL) of the soybean PC was administered once daily via themouse tail vein. On the day following the final administration (day 14post-inoculation), the lymphocyte suspension (5×10⁶ cells) wasadministered via the tail vein. The tumors were harvested from the miceat 1 hour after the lymphocyte administration.

(1-5) Preparation of Tumor Tissue Specimens and Examination ofIntratumoral Lymphocyte Infiltration

Tumor tissue specimens were prepared in the same manner as in Example 1.Lymphocyte intratumoral infiltration was examined in the same manner asin Example 1 except that an anti-EGFP antibody (MBL Life science) wasused as the antibody.

(2) Results

The results are shown in FIG. 12. The control group showed littleinfiltration of the intravenously administered lymphocytes into thetumor. In contrast, the soybean PC group showed intratumoralinfiltration of the intravenously administered lymphocytes.

Example 7: Combined Effect of Phosphatidylcholine and Immune CellInfusion Therapy on Melanoma

Intratumoral infiltration of lymphocytes administered intravenously wasexamined in the same manner as in Example 6 except that B16 cells (mousemelanoma cell line) and C57BL/6NCrSlc mice were used.

The results are shown in FIG. 13. Similarly to the results of Example 6,the control group showed little infiltration of the intravenouslyadministered lymphocytes into the tumor, whereas the soybean PC groupshowed intratumoral infiltration of the intravenously administeredlymphocytes.

Example 8: Combined Effect of Phosphatidylcholine and Immune CellInfusion Therapy on Lewis Lung Cancer

Intratumoral infiltration of lymphocytes administered intravenously wasexamined in the same manner as in Example 6 except that LLC cells (Lewislung cancer cell line) and C57BL/6NCrSlc mice were used.

The results are shown in FIG. 14. Similarly to the results of Example 6,the control group showed little infiltration of the intravenouslyadministered lymphocytes into the tumor, whereas the soybean PC groupshowed intratumoral infiltration of the intravenously administeredlymphocytes.

The results of Examples 6, 7 and 8 demonstrate that PC-mediatedpreconditioning of intratumoral vessels to facilitate lymphocyteinfiltration can enhance the antitumor effect of the intravenouslyadministered lymphocytes. Moreover, since such enhancement of theantitumor effect was observed similarly in different types of cancersand different strains of mice, PC is expected to enhance the efficacy oftumor immunotherapy in human populations with different immune statusand different genetic backgrounds. In recent years, as a therapeuticapproach to enhance antitumor immunity, administration of lymphocytesobtained by induced differentiation of iPS cells, ES cells orhematopoietic stem cells has been suggested. Also in such a therapeuticapproach, which uses lymphocytes derived from undifferentiated cells, itis considered useful to precondition tumor vessels with the use of PC.

Example 9: Examination of Effects of Various Phosphatidylcholines

As is known in the art, soybean PC is a heterogenous mixture ofphosphatidylcholines having fatty acids of different lengths and degreesof unsaturation. In this study, in order to examine whether any PC wouldproduce the same effects as those of soybean PC, various PCs were testedin cancer-bearing mice.

The PCs were classified into the following two groups (see Table 1).

Group A: PCs in which the two fatty acids are the same.Group B: PCs in which the two fatty acids are different from each other.

The PCs of Group A were dioctanoyl phosphatidylcholine (Avanti POLARLIPIDS), dimyristoyl phosphatidylcholine (Nippon Fine Chemical),distearoyl phosphatidylcholine (Nippon Fine Chemical), dioleoylphosphatidylcholine (Nippon Fine Chemical) and dilinoleoylphosphatidylcholine (Avanti POLAR LIPIDS). The PCs of Group B were(1-palmitoyl-2-myristoyl) phosphatidylcholine (Avanti POLAR LIPIDS) and(1-palmitoyl-2-oleoyl) phosphatidylcholine (Avanti POLAR LIPIDS).

In the same manner as in Example 1, LLC cells (1×10^(b) cells in 100 μLof PBS per animal) were subcutaneously injected into C57BL/6NCrSlc miceaged 8 weeks (females, SLC, Inc.) to generate cancer-bearing mice. For 7consecutive days from day 7 to day 13 post-cancer cell inoculation, thePCs indicated above were administered at a dose of 3 mg/kg (100 μL) oncedaily via the mouse tail vein. The vehicle was administered to the miceof the control group. On the day following the final administration (day14 post-inoculation), the tumors were harvested from the mice. In thesame manner as in Example 1, tumor tissue sections were prepared, tumorvasculature was observed, and intratumoral lymphocyte infiltration wasexamined.

The results of intratumoral lymphocyte infiltration are shown inTable 1. In all the PC administration groups, intratumoral infiltrationof both CD4-positive cells and CD8-positive cells were promoted. As arepresentative example, the results of the administration of distearoylphosphatidylcholine of Group A are shown in FIG. 15. Also in thespecimens of the other PC administration groups, similar images wereobtained. The results of the observation of tumor vasculature showedthat proliferation of blood vessels with a vein-like morphology wasobserved in all the PC administration groups. In addition, the resultsof the measurement of the tumor volume after the 7-day PC administrationshowed that tumor growth was inhibited in all the PC administrationgroups as compared with the control group.

TABLE 1 Lymphocyte Group Name of phosphatidylcholine Infiltration ADioctanoyl phosphatidylcholine yes Dimyristoyl phosphatidylcholine yesDistearoyl phosphatidylcholine yes Dioleoyl phosphatidylcholine yesDilinoleoyl phosphatidylcholine yes B (1-Palmitoyl-2-myristoyl)phosphatidylcholine yes (1-Palmitoyl-2-oleoyl) phosphatidylcholine yes

Example 10: Improving Effect of Phosphatidylcholine on IntratumoralHypoxia

LLC cells (1×10⁶ cells in 100 μL of PBS per animal) were subcutaneouslyinjected into C57BL/6NCrSlc mice aged 8 weeks (females, SLC, Inc.) togenerate cancer-bearing mice. In the same manner as in Example 1,soybean PC or Intralipos was administered once daily via the mouse tailvein for 7 consecutive days from day 7 to day 13 post-cancer cellinoculation. The vehicle was administered to the mice of the controlgroup. On day 14 post-tumor cell inoculation, pimonidazole (Hypoxyprobe,Burlington, Mass., USA) was intraperitoneally administered at a dose of60 mg/kg (100 ILL), and 2 hours later, the tumors were harvested fromthe mice. Tumor tissue sections were prepared in the same manner as inExample 1, and the hypoxic area was visualized with an anti-pimonidazoleantibody.

The results are shown in FIG. 16. As is clear from FIG. 16, the hypoxicarea was significantly diminished in the soybean PC group and theIntralipos group, as compared with the control group. This shows theimprovement of intratumoral blood flow by PC administration. Sincehypoxia in cancerous tissue is considered to cause mutations in thechromosomal DNAs of cancer cells and to induce malignant transformationof cancer cells, the improvement of blood flow by PC is expected topotentially inhibit malignant transformation of cancer cells.

Example 11: Promoting Effect of Phosphatidylcholine on Intratumoral DrugDelivery

LLC cells (1×10⁶ cells in 100 μL of PBS per animal) were subcutaneouslyinjected into C57BL/6NCrSlc mice aged 8 weeks (females, SLC, Inc.) togenerate cancer-bearing mice. In the same manner as in Example 1,soybean PC or Intralipos was administered once daily via the mouse tailvein for 7 consecutive days from day 7 to day 13 post-cancer cellinoculation. Nothing was administered to the mice of the control group.On day 14 post-tumor cell inoculation, doxorubicin (Nippon Kayaku Co.,Ltd.), which is an anticancer drug emitting red fluorescence, wasintravenously administered at a dose of 3 mg/100 μL, and 20 minuteslater, the tumors were harvested from the mice. Tumor tissue sectionswere prepared in the same manner as in Example 1 and observed for thered fluorescent substance (doxorubicin) in the tumors.

The results are shown in FIG. 17. As is clear from FIG. 17, the controlgroup showed little intratumoral penetration of doxorubicin, but in thesoybean PC group and the Intralipos group, doxorubicin was delivered tothe central region of the tumor.

Example 12: Antitumor Effect of Combination of Phosphatidylcholine and5-FU

The above studies confirmed that PC administration improves intratumoralblood flow, thereby achieving efficient drug delivery to the deep partof the tumor. In this study, the antitumor effect of a combination of PCand an anticancer drug was examined.

LLC cells (1×10⁶ cells in 100 μL of PBS per animal) were subcutaneouslyinjected into C57BL/6NCrSlc mice aged 8 weeks (females, SLC, Inc.) togenerate cancer-bearing mice. The mice were assigned to 4 groups: asoybean PC group, a 5-FU group, a soybean PC plus 5-FU group, and acontrol group (vehicle administration group) (3 animals per group). Forthe soybean PC group and the soybean PC plus 5-FU group, in the samemanner as in Example 1, 3 mg/kg (100 μL) of the soybean PC wasadministered once daily via the mouse tail vein for 13 consecutive daysfrom day 7 to day 20 post-cancer cell inoculation. For the 5-FU groupand the soybean PC plus 5-FU group, 5-FU (Kyowa Hakko Kirin Co., Ltd.)was intraperitoneally administered at a dose of 20 mg/kg on day 7 andday 14 post-cancer cell inoculation. Tumor volume was measured on day 7,day 14 and day 21 post-cancer cell inoculation. The tumor volume wascalculated by the following formula: length×width×height×0.5.

The results are shown in FIG. 18. The soybean PC group and the 5-FUgroup showed tumor growth inhibition as compared with the control group,and the soybean PC plus 5-FU group showed more remarkable tumor growthinhibition than that observed in the groups having received either thesoybean PC or 5-FU alone.

The above results show the following: PC enlarges the diameter ofintratumoral vessels and promotes vein-like morphological change ofintratumoral vessels; PC also induces the connection of intratumoralvessels, resulting in an improved blood flow; and these effects mediatethe promotion of drug delivery into tumor tissue, thus remarkablyenhancing the effect of the anticancer drug.

Example 13: Effect of Soybean Phosphatidylcholine on HUVEC

The above studies were performed to analyze the effects of PC on bloodvessels in cancer-bearing mouse models. In this study, the effect of PCon human vascular endothelial cells were analyzed.

(1) Experimental Method

Each well of a 48-well plate was coated with 200 μL of Matrigel (BDbiosciences), and human umbilical vein endothelial cells (HUVECs; KuraboIndustries Ltd.) were seeded at 5×10⁴ cells/200 μL/well and cultured.For the culture, HuMedia-EB2 medium (Kurabo Industries, Ltd.)supplemented with 1% FCS was used. VEGF (recombinant human VEGF165,Peprotech) was added at a final concentration of 10 ng/mL to the culturemedium to induce tubulogenesis of the vascular endothelial cells.Soybean PC was also added at 10 μM concomitantly with VEGF. At 12 to 16hours after the start of the culture, the cells were photographed undera microscope. In addition, Hoechst (Boehringer Ingelheim) was added tostain cell nuclei, and newly formed blood vessels were observed. Thewells not containing the soybean PC were used as a control.

(2) Results

The results are shown in FIG. 19. The control group and the soybean PCgroup showed induced tubulogenesis, and in the soybean PC group, someblood vessels had an enlarged diameter (shown by the arrows). Thestudies of the cancer-bearing mouse models in the above Examplesconfirmed that PC administration enlarges the diameter of intratumoralvessels and promotes vein-like morphological change of intratumoralvessels. The results of this Example show that PC administrationpromotes vein-like morphological change of human blood vessels as well.

Example 14: Effect of Various Phosphatidylcholines on HUVEC

The same experiment as in Example 12 was performed using soybean PC andother 8 types of PCs. The PCs were classified as Group A or B as withExample 8. The PCs used were as follows (see Table 2).

Group A:

(1) dioctanoyl phosphatidylcholine (Avanti POLAR LIPIDS)(2) dimyristoyl phosphatidylcholine (Nippon Fine Chemical)(3) distearoyl phosphatidylcholine (Nippon Fine Chemical)(4) dioleoyl phosphatidylcholine (Nippon Fine Chemical)(5) dilinoleoyl phosphatidylcholine (Avanti POLAR LIPIDS)(6) didocosahexaenoyl phosphatidylcholine (Avanti POLAR LIPIDS)

Group B:

(7) (1-palmitoyl-2-myristoyl) phosphatidylcholine (Avanti POLAR LIPIDS)(8) (1-palmitoyl-2-oleoyl) phosphatidylcholine (Avanti POLAR LIPIDS)

The results are shown in Table 2 and FIG. 20. The control group, thesoybean PC group and the other PC groups (1 to 8) all showed inducedtubulogenesis, and in the soybean PC group and the other PC groups (1 to8), some blood vessels had an enlarged diameter (shown by the arrows).That is, the results show that not only soybean PC but other PCsindicated above are effective for promoting vein-like morphologicalchange of vessels.

TABLE 2 Vein-like morphological Group Name of phosphatidylcholine changeA Dioctanoyl phosphatidylcholine yes Dimyristoyl phosphatidylcholine yesDistearoyl phosphatidylcholine yes Dioleoyl phosphatidylcholine yesDilinoleoyl phosphatidylcholine yes Didocosahexaenoylphosphatidylcholine yes B (1-Palmitoyl-2-myristoyl) phosphatidylcholineyes (1-Palmitoyl-2-oleoyl) phosphatidylcholine yes

The present disclosure is not limited to the embodiments and examplesdescribed above, and various modifications can be made within the scopeof the appended claims. Other embodiments provided by suitably combiningtechnical means disclosed in separate embodiments of the presentdisclosure are also within the technical scope of the presentdisclosure. All the academic publications and patent literature cited inthe description are incorporated herein by reference.

1. A vein-formation promoting agent comprising a phosphatidylcholine as an active ingredient, the agent being capable of promoting vein-like morphological change of tumor vessels.
 2. The vein-formation promoting agent according to claim 1, wherein the agent is capable of enlarging the diameter of tumor vessels and/or promoting connection of tumor vessels to each other.
 3. The vein-formation promoting agent according to claim 1, wherein the phosphatidylcholine is one kind of phosphatidylcholine or a mixture of two or more kinds of phosphatidylcholines.
 4. The vein-formation promoting agent according to claim 1, wherein the agent is used in combination with cancer immunotherapy.
 5. The vein-formation promoting agent according to claim 4, wherein the cancer immunotherapy is a therapy for reversal of immunosuppression and/or immune cell infusion therapy.
 6. The vein-formation promoting agent according to claim 5, wherein the therapy for reversal of immunosuppression uses an immune checkpoint inhibitor, and wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, a PD-L1 blocker or an anti-PD-L1 antibody.
 7. A vessel-diameter enlarging agent comprising a phosphatidylcholine as an active ingredient, the agent being capable of enlarging the diameter of tumor vessels.
 8. A blood vessel-connection promoting agent comprising a phosphatidylcholine as an active ingredient, the agent being capable of promoting connection of tumor vessels to each other without mediation of a lysophospholipid receptor.
 9. A leukocyte-infiltration promoting agent comprising a phosphatidylcholine as an active ingredient, the agent being capable of promoting infiltration of leukocytes throughout a tumor region without mediation of a lysophospholipid receptor.
 10. The leukocyte-infiltration promoting agent according to claim 9, wherein the leukocytes are CD4-positive cells and/or CD8-positive cells.
 11. An antitumor immunostimulatory agent comprising a phosphatidylcholine as an active ingredient, the agent being capable of promoting infiltration of leukocytes throughout a tumor region without mediation of a lysophospholipid receptor.
 12. The antitumor immunostimulatory agent according to claim 11, wherein the leukocytes are CD4-positive cells and/or CD8-positive cells. 